Function Analysis Of A Stress-responsive Transcription Factor Gene GhSNAC3 In Gossypium Hirsutum L.

China is an important trading country of cotton, cotton occupies an important position in the national economy of our country. However in recent years, cotton production area is being transferred to lands of salt, drought or other barren soils. Therefore it is of great significance to explore the cotton genes involved in stress responses. NAC transcription factors belong to a unique class of transcription factors in plants, which are also one of the largest transcription factor families in plant genomes. Studies showed that NAC transcription factors have multiple biological functions, widely involved in plant growth and development adjustment, tissue morphogenesis, plant hormones signal transduction, and plant responses to both biotic and abiotic stresses.Object: To explore the NAC gene resources of upland cotton, the NAC gene was identified and clone. Through the study of Gh NAC gene expression, transcription and gene transfer, we preliminarily determined the function of Gh NAC gene in response to adversity stress in cotton. And the function of Gh NAC gene was further verified by VIGS technology and promoter analysis.Methods: The potential NAC genes in cotton were predicted by bioinformatics methods; Gh SNAC3 was isolated from cotton via RT-PCR; The expression patterns of Gh SNAC3 under various stress conditions was studied using fluorescence quantitative RT-PCR; A fusion protein gene containing Gh SNAC3 and GFP was constructed using the p BI221-GFP vector, for transient expression analysis by laser scanning confocal microscopy. The yeast transcriptional activator vector constructed using p CAMBIA3301 and p ROK Ⅱ, and transformed into tobacco. Furthermore, the Gh SNAC3 gene silenced plants were obtained through infecting coton leaves with a virus induced gene silencing carrier(VIGS) constructed using TRV2 plasmid.We cloned the 5’flanking regulatory sequence of Gh SNAC3 gene, and analyzed itsregulatory elements using online software.Results:(1)The transcription factor gene Gh SNAC3 was cloned, with a full length c DNA of 1072 bp, encoding a protein of 276 amino acids. Gene structure analysis of Gh SNAC3 showed that it has three exons and two introns which has the characteristic feature of plant introns. The Gh SNAC3 protein contains the NAM domain at its N-end.(2)Transient expression analysis of the fusion gene showed that the Gh SNAC3 protein was located in the necleus; Construction of yeast transcriptional activation carrier and yeast transformation, the results showed that the tansformed yeast growed normally on SD medium and the collony color was blue when the medium contained β-gal and IPTG, indicating Gh SNAC3 has transcriptional activation activity.(3)The q RT-PCR analysis revealed that the expression of Gh SNAC3 responds to drought, high salinity and verticillium wilt infection stresses in varying degrees.(4)And the over-expression vector transformed into tobacco, the transgenic plants showed improved resistances to salinity and drought stresses. Furthermore, the Gh SNAC3 gene silenced plants were obtained through infecting coton leaves with a virus induced gene silencing carrier(VIGS), compared to the control group, these plants showed weakened resistance to drought and high salinity, and some of which appeared delayed maturity.(5)In addition, the Gh SNAC3 promoter 1279 bp was cloned and analyzed. It contained several cis-acting elements associated with plant growth and development, and plant responses to adversity stresses.(6)And the expression vector transformed into tobacco, the transgenic plants showed improved resistances to stress. In addition to the semi-quantitative PCR technique showed that Gh SNAC3 expression has tissue-specific.Conclusion: In this study, we have cloned the NAC transcription factor and promter of gene Gh SNAC3 from Gossypium hirsutum L. Gh SNAC3 played an important role in the resistance responses to various adversity stresses in cotton, such as drought, high salt and Verticillium Wilt. The results provided the genetic resources for the improvement of cotton breeding. Moreover Gh SNAC3 promoter is a stress inducible promoter, and with various expression levels.